Drug abuse represents an aggravating factor in the treatment of HIV/AIDS, which is associated with higher viral load, faster disease progression, and enhanced transmission. Understanding the mechanisms of these potentiating effects at the molecular level could lead to significant changes in the administration of current anti-retroviral treatments and the discovery of new therapeutic targets. Recent findings on the activity of N6-methyladenosine (m6A) in HIV-1 replication and its accumulation in cocaine- treated cells suggest that RNA post-transcriptional modification (PTM) may represent a key component of extensive communications between drug and RNA processes. In addition to m6A, the implementation of a comprehensive analytical approach based on mass spectrometry (MS) allowed us to identify numerous other PTMs on viral RNA isolated from infected cells and virions. Their striking diversity and abundance support the proposal?s hypothesis that RNA PTMs may mediate virus-host communications by modulating essential molecular interactions, which can be influenced by neuromodulators intracellular signaling. Our highly innovative approach will allow us to test this hypothesis by identifying RNA processes that are directly affected by the presence of PTMs, determining the effects of dopamine and GABA signal transduction on PTM expression, and finally recognizing overlaps between the respective pathways, which will reveal possible crosstalk mechanisms between intracellular signaling and viral infection. To this effect, we will perform silencing of PTM enzymes and key regulatory genes to retrace their control networks, while simultaneously monitoring the PTM landscape and essential indicators of virus and cell fitness. We will determine the variations of PTM landscapes in the absence and presence of dopamine and GABA, while simultaneously monitoring the effects on prominent RNA processing infrastructure. We will then probe the PTMs that displayed the most significant variations during both infection and neuromodulator treatment. The fact that the HIV-1 genome does not code for any known biogenetic enzyme implies that host enzymes must be responsible for the detected viral PTMs, thus placing them under the control of key intracellular signaling. Modification may represent adaptation to the host?s metabolic infrastructure, which enables the viral RNA to evade surveillance and elimination by the host?s defenses. Therefore, it is possible to envision that this putative adaptation mechanism could be affected by neuromodulator signaling pathways. This project will look for evidence and will establish the framework necessary to guide in-depth mechanistic investigations. The transformative impact of the proposed activities will be substantiated by the ability to test this hypothesis in comprehensive fashion, expose the influence of PTMs on prominent RNA processes, identify their possible regulatory mechanisms, and understand the significance of their biogenetic enzymes in viral lifecycle and fitness.